While mechanisms regulating oncogenesis have been elucidated substantially in recent years, comparatively little is yet known about the mechanisms that underlie metastasis of tumor cells, the ultimate cause of death in cancer. Metastasis suppressor genes play pivotal roles in controlling this process, but only a handful have been identified. NM23 ("negative in metastasis") genes represent the first described metastasis suppressors and are recognized for their antimetastatic activity in melanoma, breast and gastric carcinoma. The overall goal of this project to systematically dissect the molecular mechanisms and genomic pathways underlying the metastasis suppressor activity of NM23. We recently found that the NM23-H1 isoform is a 3'-5'exonuclease (3'5'EXO), an activity often associated with genome integrity, apoptosis and chromatin remodeling. Although there is clear potential relevance of these processes to malignant progression, we will also measure the extent to which the nucleoside diphosphate kinase (NDPK) and histidine kinase (hisK) activities of NM23-H1 contribute to its metastasis suppressor properties. In Specific Aim 1, we will complete a detailed structure-function analysis to delineate the active site of the 3'5 EXO, and characterize a panel of mutants harboring lesions that selectively disrupt the 3'5'EXO, NDPK and hisK functions of the NM23-H1 molecule. The mutants will be used as molecular tools to determine the relative contributions of each to suppression of the metastatic phenotype (Specific Aim 2) in human melanoma cells, using cell culture and in vivo models to measure tumorigenic and metastatic potential. Gene expression changes elicited by expression of NM23-H1 variants will be determined by microarray analysis and real-time PCR (Specific Aim 3). Correlational analysis will identify NM23-regulated genes whose expression tracks with metastatic phenotype and metastatic progression. Functional relevance of identified genes will be validated by viral-mediated overexpression and knockdown approaches. In Specific Aim 4, we will directly test the DMA repair activity of NM23-H1 in mammalian and yeast cell models, and identify the relevant enzymatic function. These studies should provide the first systematic analysis of pathways through which NM23 proteins oppose metastasis in multiple cancer types and, therefore could suggest novel strategies to combat this final and intractable stage of the disease.